1. Field of the Invention
The invention relates to the fields of oncology and diagnostic testing. The invention is useful for screening, staging, treatment responses, recurrence or the like in diseases such as cancer or cardiovascular disorders. More specifically, the present invention provides methods which facilitate analysis and enumeration of circulating rare cells isolated from biological samples.
2. Background
Methods for the characterization of not only tumor cells, but also rare cells, or other biological entities from biological samples have been previously described (U.S. Pat. No. 6,365,362). This two stage method requires efficient enrichment to ensure acquisition of target cells while eliminating a substantial amount of debris and other interfering substances prior to analysis, allowing for cellular examination by imaging techniques. The method combines elements of immunomagnetic enrichment with multi-parameter flow cytometry, microscopy and immunocytochemical analysis in a uniquely automated way. The combination method is used to enrich and enumerate epithelial cells in blood samples, thus providing a tool for measuring cancer.
The two stage method has applications in cancer prognosis and survival for patients with metastatic cancer (WO 04076643). Based on the presence of morphologically intact circulating cancer cells in blood, this method is able to correlate the presence of circulating cancer cells of metastatic breast cancer patients with time to disease progression and survival. More specifically, the presence of five (5) or more circulating tumor cells per 7.5 milliliters provides a predictive value at the first follow-up, thus providing an early prognostic indicator of patient survival.
The specificity of the assay described above increases with the number of cells detected and is not sufficient in cases were only few (generally less than 5 circulating tumor cells) are detected. One solution to this problem is to provide detailed genetic information about suspected cancer cells. Accordingly, a method that would incorporate enrichment of a blood sample with multi-parametric image cytometry and multi-parametric genetic analysis on an individual suspect cancer cell would provide a complete profile and confirmatory mechanism to significantly improve current procedures for patient screening, assessing recurrence of disease, or overall survival. A confirmatory assay in the analysis of rare circulating cells by combining phenotypic and genotypic multiparametic analysis of an individually isolated target cell has been described (see pending U.S. application Ser. No. 12/067,532). Confirmation provides for a clinically significant level of sensitivity and, therefore, assurance to the clinician of any quantitative information acquired. Relevant disease states are assessed using extremely small (1, 2, 3, or 4) numbers of circulating tumor cells (CTC's) and provide a confirmation for early disease detection.
There are no other technologies available that can perform multiple high sensitivity assays on the same sample with the same marker and do it on rare events. Multiparameter flow cytometry is commonly done, but it requires hundreds or thousands of target cells or events to get accurate information. However if a patient only has 6 CTCs in 7.5 mLs of blood there are too few events to even detect reliably, to say nothing of performing multiparameter analysis.
The present invention extends the enrichment and analysis protocol described in U.S. Pat. No. 6,365,362 and utilized in Celltracks® Autoprep® System and Celltracks® Analyzer II System (Immunicon Corporation, Huntingdon Valley, Pa.) by providing a means to allow for the interrogation of rare circulating cells with multiple fluorescent biomarkers